(i) Field of the Invention
This present invention relates to a method for detecting a chemical transformation of a substrate such that said chemical transformation of this substrate releases a product, whereby said released product is then a detectable product.
The invention also relates to substrates that can be used in this method. The invention also relates to the use of this method in a process for detection and optionally quantification of a chemical transformation in a sample.
(ii) Description of Related Art
After reaction, many substrates release a product that provides an easily identifiable signal. It is possible to cite the well known examples of glycosides, esters and phosphates of phenols whose hydrolysis directly releases phenol, which is detected in general by its color (chromogenic substrate) or its fluorescence (fluorogenic substrate)) (D. C. Demirjian, P. C. Shah, F. Moris-Varas, Top. Curr. Chem. 1999, 200, 1; M. T. Reetz, K.-E. Jaeger, Top. Curr. Chem. 1999, 200, 31).
It is also possible to cite 2-methoxy-1-naphthalene methanol, whose oxidation provides fluorescent 2-methoxy-1-naphthaldehyde (B. L. Vallee, U.S. Pat. No. 5,162,203; B. List, C. F. Barbas, R. A. Lerner, Proc. Natl. Acad. Sci. USA 1998, 95, 15351 for use in aldolases).
These types of substrate have a major disadvantage: the chromogenic or fluorogenic phenol is a strongly activated group, which makes these substrates unstable and susceptible to non-specific reactions. In the case of 2-methoxy-1-naphthalene methanol, the problem is similar since the benzylic position is sensitive to non-specific oxidation.
A second class of substrate leads to the revealing of a product that is obtained after enzymatic secondary reaction and/or spontaneous secondary reaction. Several examples exist, such as penicillinase substrates (U.S. Pat. No. 5,583,217), alcohol dehydrogenase (ADH) substrates (G. Klein, J.-L. Reymond, Bioorg. Med. Chem. Lett. 1998, 8, 1113) and aldolase substrates (N. Jourdain, R. Pérez Carlón, J.-L. Reymond, Tetrahedron Lett. 1998, 39, 9415).
It is also possible to note the existence of a substrate for measuring the NADH (C. A. Roeschlaub, N. L. Maidwell, M. R. Rezai, P. G. Sammes, Chem. Commun. 1999, 1637).
Finally, it is possible to cite substrates whose detected product of the secondary reaction is obtained by the action of the beta-galactosidase (K. L. Matta, C. F. Piskorz, J. J. Barlow, Carbohydr. Res. 1981, 90, C1-C3) or by ADH (G. Klein, J.-L. Reymond, Helv. Chim. Acta 1999, 82, 400).
This second class of substrate is more stable. This second class of substrate, however, is limited to particular uses, because the fact of using an enzyme for the secondary reaction represents a drawback in terms of cost and flexibility.
The research work carried out within the scope of this invention consisted in particular in developing a method for detecting a chemical transformation. Thus, this invention proposes solving the problems reported above by using a method for measuring a chemical transformation that is reliable, sensitive and reproducible, using a stable substrate within the environment in which the chemical reaction occurs, and such that the successive transformations release a detectable product.